Prostate-specific antigen (PSA) is a 33 to 34 kDa glycoprotein produced primarily by prostate epithelium, and is known as the most common serum marker for diagnosing prostate cancer. Prostate cancer usually leads to the release of high concentrations of PSA into the circulatory system and causes serum PSA levels to rise up to 105-fold. Thus, measurement of serum PSA is widely used for early detection and monitoring of patients with prostate cancer. A serum PSA measurement above a cut-off value of 4.0 ng/mL is generally regarded as positive and might indicate the need for a biopsy.
Aptamers are nucleic acid ligands that can recognize various target molecules, such as proteins and small molecules, with high affinity and specificity comparable to those of monoclonal antibodies. Aptamers, especially DNA aptamers, are easily and inexpensively synthesized and chemically modified. Furthermore, they can be designed to undergo structural changes when they bind to targets. These features make DNA aptamers ideal for molecular recognition elements in biosensors. Using the advantages of aptamers, aptamer-based highly sensitive detection systems have been constructed.
Target-binding ability is the most important feature in terms of applying an aptamer to a biosensor. Aptamers are generally selected from random sequence pools in vitro using a process referred to as SELEX (systematic evolution of ligands by exponential enrichment) based on target-binding activity. SELEX is an efficient screening method because iterative cycles of selection can be carried out using Polymerase Chain Reaction (PCR).
Under these circumstances, various aptamers are known as detection tools for, for example, prion specific proteins (JP No. 2006-42645 A 1), vascular endothelial growth factors (JP 2008-237042) and insulin (JP No, 2009-183192 A1).
In particular, WO2006/096754 describes stabilized aptamers capable of binding to prostate-specific membrane antigen (PSMA) and their use as prostate cancer therapeutics, and discloses that aptamers obtained by minimization and optimization have Kd (dissociation constant)=2 to 10 (nM).
On the other hand, it is known that SELEX sometimes fails to screen for aptamers with high affinity to target molecules and that the actual size and complexity of the sequence space in which aptamers are screened by SELEX is smaller than expected. In order to obtain an aptamer having higher affinity, various modification methods of screening by SELEX are known, such as a selection method for DNA aptamer using genetic algorithms (GAs) (see, for example, Nucleic Acids Res., 2005, Vol. 33 (12), e108, JP No. 2007-14292 A). For the selection of aptamer using GA based on target-binding ability, candidate oligonucleotides are first pre-selected by SELEX. Their oligonucleotide sequences are then amplified, crossed over and mutated in silico using GA. After the GA operations are performed, a new set of sequences is synthesized and assayed in vitro. Then, sequences with high binding ability are selected for a next cycle of GA. By repeating the process of GA operation, the sequence space, which cannot be fully screened only by SELEX, can be covered.
However, because the binding ability of an aptamer is generally lower than that of an antibody, aptamers are not generally practical as diagnosis tools, and, for example, the PSMA binding aptamers described above are not sufficient for diagnosis of prostate cancer for the same reason.